Norman R. Joseph and William C. Stadie J. Biol. Chem. 1938, 125:795-799. Access the most updated version of this article at http://www.jbc.org/content/125/2/795.citation Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites . Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/125/2/795.citation.full.h tml#ref-list-1 Downloaded from http://www.jbc.org/ by guest on October 6, 2014 ARTICLE: THE SIMULTANEOUS DETERMINATION OF TOTAL BASE AND CHLORIDE ON THE SAME SAMPLE OF SERUM BY ELECTRODIALYSIS THE SIMULTANEOUS AND CHLORIDE SERUM BY (Prom NORMAN R. the John Herr University (Received DETERMINATION OF TOTAL BASE ON THE SAME SAMPLE OF BY ELECTRODIALYSIS JOSEPH AND WILLIAM Musser Department of Pennsylvania, for publication, C. STADIE oj Kesearch Philadelphia) July Medicine, 22, 1938) 705 Downloaded from http://www.jbc.org/ by guest on October 6, 2014 A11 electrodialytic method for the determination of total base in serum and other biological fluids has been recently described by Keys (5), whose procedure is based on the earlier work of Stoddard (7) and Adair and Keys (1). Mercury within a cylindrical cathode vessel is separated from the material to be analyzed by a cellophane membrane. As the electric current is transported, the diffusible cations form an amalgam, which is allowed to react with standard acid. Upon completion of the electrodialysis, the acid within the cathode vessel is titrated with standard alkali. The total base in the sample is estimated as equivalent to the displaced hydrogen. Determination of total base by this method is rapid and eliminates the ashing required in the Fiske (3) and Stadie-Ross (6) methods. A modification of the Adair-Keys method is here presented by which the total base and chloride are simultaneously determined An anode chamber, separated by in t)hc sa.me sample of serum. a cellophane membrane from the middle chamber cont’aining the sermll, is addrd to the system. After electrodialysis, the total base in the cathode chamber is determined as in the Adair-Keys method, and the chloride, collected in the anode chamber free of protein, is determined by the Volhard method (10). The successful quantitative determination of chloride by electrodialysis depends upon the use, in the anode chamber, of a reducing agent to prevent the oxidation of chloride ion into free chlorine which would escape Control analyses showed that, in the absence of such a reducing agent,, up to 50 per cent of t,he 796 Total Base and Chloride chloride might be lost. The reducing agent completely prevents loss of Cl- and 100 per cent recovery is possible. It was found that glucose in dilute acetic acid served admirably as the reducing agent to be used in the anode chamber. A somewhat similar method of electrodialysis has been employed by di Benedetto (2), who was able, however, to effect only partial recovery of base. Apparatus and Procedure Downloaded from http://www.jbc.org/ by guest on October 6, 2014 The apparatus employed is illustrated in Fig. 1. It consists essentially of three vessels, a beaker (A), about 13 inches in diameter, which serves as the anode vessel, and two concentric cylinders (B and C), respectively about $ inch and $ inch in diameter. The latter are arranged as in the illustration, the larger cylinder supported in the beaker by wire attached near the upper end, while the smaller cylinder can be suspended in the larger by the same means. The anode solution contains dilute acetic acid, as electrolyte, and glucose, added to reduce any free chlorine at the anode. The larger cylinder contains the material to be analyzed, while the smaller one is the cathode vessel. The lower end of each cylinder is enclosed by a cellophane membrane. A square piece of du Pont’s No. 300 cellophane is moistened in water for a few minutes, then dried, and stretched tightly over the end of the cylinder. After the membrane is held firmly in place for a short time, the upper edges are trimmed with a sharp knife or scalpel, and are fixed to the cylinder by means of collodion. An electrodialysis is carried out in the following manner. Approximately 5 cc. of electrolyte solution are introduced into the anode vessel. We have used 0.05 N acetic acid plus 3 per cent glucose for this purpose. Approximately 0.5 cc. of purified mercury is introduced into the cathode vessel and covered with 2 cc. of 0.1 N hydrochloric acid. A platinum cathode makes contact with the mercury. 0.5 cc. of serum or unknown solution is pipetted into the larger cylinder, and is diluted with about 5 cc. of water. The concentric cylinders are then suspended in the anode vessel, a platinum anode is inserted into the anode solution, and electrolysis is permitted to proceed. 110 volts direct current N. R. Joseph and W. C. Stadie 797 are used, passing through an external resistance of about 1000 ohms. The electrodialysis is carried out for about 1 hour, at the end of which time the anode and cathode vessels are removed and their contents titrated. The anode solution, as well as the cathode solution, is protein-free, and chloride can be determined without the necessity of ashing. CATHODE (PLATINUM) MERCURY o FIG. 1. Electrodialysis ANODE: PLAT\NUM FOIL: (4mm’) apparatus Analysis Total base is determined by titration of the contents of the cathode vessel with standard alkali, a burette graduated to 0.05 cc. being employed. A convenient concentration of standard alkali is 0.025 N. The total base expressed in milliequivalents per liter of serum is given by the expression Total base = ‘g (0.2 - 0.025 X cc. NaOH) Downloaded from http://www.jbc.org/ by guest on October 6, 2014 CATHODE Total Base and Chloride Chloride is determined by%itrating the contents of the anode vessel according to the Volhard method (10). It is precipitated as silver chloride by the addition of 1 cc. of 0.15 N silver nitrate. After removal of the precipitate by filtration the excess silver is with ferric alum as titrated with 0.02 N potassium thiocyanate, TABLE Comparison 01 Total -, - Igo. of determinations _- .- Base Chloride 16 154.5 f 0.7 104.3 f 0.3 6 6 152.7 f 0.7 205.2 f 0.7 104.5 zk 0.2 154.2 f 0.4 B 12 204.5 f 0.7 151.6f0.8 154.3 f 0.3 103.0 f 0.5 A 6 16 149.5 f: 0.7 162.0 f 0.6 103.0 f: 0.4 107.0 * 0.4 16 Base 6 Chloride 161.0f0.7 106.8 f 0.4 A + 0.5 cc. 0.05 iv NaCl _- Method Electrodialysis Ashing* Electrodialysis Calculated Electrodialysis Ashing* Eleetrodialysis Ashingt * Base was determined by the Stadie-Ross (6) procedure on 0.5 cc. samples; chlorides by the Wilson-Ball (11) method on 0.5 cc. samples. t Base was determined as in the Stadie-Ross procedure, except that 15 cc. samples were analyzed. indicator. Chloride expressed as milliequivalents calculated from the equation Chloride = ‘2 per liter is (0.15 - 0.02 X cc. KCKS) Results The results of a number of analyses of serum are given in Table I, and are compared with analyses according to standard methods. The Stadie-Ross (6) procedure was employed for base, and chlo- Downloaded from http://www.jbc.org/ by guest on October 6, 2014 The figures represent series of determinations. per liter. I Base and Chloride As Determined ~IJ Independent Methods the mean values and standard deviations of a The results are expressed in milliequivalents N. It. Joseph and w’. C. Stadie 799 SUMMARY A modification of the Adair-Keys electrodialytic method for total base is described by which both total base and chloride of serum are simultaneously determined on the same sample. BIBLIOGRAPHY 1. 2. 3. 4. 5. 6. 7. 8. 9. Adair, G. S., and Keys, A. B., J. Physiol., 81, lG2 (1934). di Benedetto, E., Rev. sot. argentina biol., 8, 497 (1932). Fiske, C. H., J. Biol. Chem., 61,55 (1922). Hald, P. M., J. Biol. Chem., 103,471 (1933). Keys, A., J. Biol. Chem., 114, 449 (1936). Stadie, W. C., and Ross, E. C., J. Biol. Chem., 66,735 (1926). Stoddard, J. Id., J. Hiol. Chem., 74, 677 (1927). Van Slyke, D. D., J. BioZ. Chem., 68, 523 (1923-24). Van Slyke, D. D., Hiller, A., and Berthelsen, K. C., J. BioZ. 659 (1927). 10. Volhard, J., Z. anal. Chem., 1’7, 482 (1878). 11. Wilson, D. \V., and Ball, E. G., J. Biol. Chefm., 79, 221 (1928). Chem., 74, Downloaded from http://www.jbc.org/ by guest on October 6, 2014 rides were determined by means of Wilson and Ball’s modification of Van Slyke’s wet ashing method (8, 11). As Table I indicates, the chloride determinations are in very good agreement with the results of the ashing m&hod. The results for total base appear to be approximately 1 per cent higher on the average t,han those obtained by the Stadie-Ross procedure. Similar results have been obtained by Consolazio (personal communication), who has compared total base determined by the Adair-Keys method wit’h that determined by Hald’s benzidine sulfate method (4). He finds the results of the electrodialytic method to be in good agreement with the gasometric method of Van Slyke, Hiller, and Berthelsen (Y), bot,h methods yielding somewhat higher values for total base than those obtained by the analysis of benzidine sulfate. The evidence thus points to a real but not serious discrepancy between the results of the two determinations. The source of this small error is as yet undetermined.